Appropriate development and antigen-dependent activation of T-cells is critically dependent upon the function of Src- and Jak- family non-receptor tyrosine kinases. The function and regulation of these kinases requires association with the cytoplasmic tails of receptors on the cell surface. Lck associates with the cytoplasmic tails of CD4 and.CDS, and Fyn associates with the tails of SLAM/CD150 family co-receptors via the adapter protein SAP. Proper signaling from the SLAM receptor is indispensable for a normal immune response; defects in the gene encoding SAP are the cause of X-linked lymphoproliferative syndrome (XLP) and other immune deficiencies. Similarly, Jak-family tyrosine kinases couple to cytokine receptors, and are activated upon engagement of their cognate receptors. The importance of Jak3 in immune function is highlighted by the fact that inactivating mutations in this kinase result in severe combined immunodeficiency (SCID). In the previous grant period, we determined three-dimensional structures that show directly how Lck and Fyn couple to the their respective receptors. Additionally, we determined the crystal structure of the kinase domain of Jak3. In this renewal, we propose to extend these studies to include larger fragments of Lck and Fyn in order to understand how receptor interactions contribute to catalytic regulation in these kinases. In Aim 1, we will determine the structure of SAP in a ternary complex with the SLAM cytoplasmic tail and a Fyn SH3-SH2-kinase fragment. Additionally, we will test our hypothesis that SAP can recruit and activate other Src kinases, including Lck. In collaboration with the Terhorst lab, we will use our structural results to dissect SLAM signaling in a SAP-deficient mouse model. In Aim 2, we use X-ray crystallography and NMR to explore how the unique domain of Lck in complex with CD4 may impinge upon the regulatory interactions among the SH3, SH2 and kinase domains of Lck. In Aim 3, we propose to study peptide/protein substrate recognition by Src and Jak kinases. These studies will directly impact health care by facilitating rational design of new drugs for use in organ transplantation and for treatment of leukemias that arise from defects in these proteins. Also, they will help us to understand the causes of immune deficiency diseases XLP and SCID at a detailed molecular level.